Annual Report 2003 This project investigates signal transduction pathways that regulate proliferation, migration, and differentiation of lens and corneal epithelial cells with the goal of identifying enzymes that may be therapeutically targeted in pathological conditions. Recent studies have focused on signaling downstream of the EGF receptor. We have previously shown that EGF-stimulated proliferation of lens epithelial cells requires endogenous synthesis of an eicosanoid, 12(S)HETE. Our recent work has focused on the mechanism of action of this compound. This year our studies have demonstrated that 12(S)HETE is responsible for translocation PKCbetaII to the lens epithelial cell membrane and have shown that this effect does not require an increase in intracellular calcium concentration, as measured by Fluo3 fluorescence. 12(S)HETE recruits PKCbetaII via its C1 domain, which contains binding sites for diacylglycerol and phospholipids, not by its C2 domain, which contains binding sites for calcium. Moreover, exogenous 12(S)HETE does not affect intracellular calcium concentration. Ongoing studies are examining the relationship of 12(S)HETE, EGF, and diaclglycerol accumulation to downstream events leading to epithelial cell proliferation. A second area of concentration seeks to determine the role of the proline-directed kinase, Cdk5, in corneal epithelial cell wound healing. EGF promotes corneal wound healing by promoting cell migration and proliferation. Through transfection studies and the use of specific Cdk5 inhibitors, we have demonstrated that Cdk5 activity inhibits migration of cultured mouse corneal epithelial cells by strengthening cell adhesion. In a recent collaboration with Dr. Mary Ann Stepp, we have used transgenic mice expressing Cdk5 or a kinase inactive mutation of Cdk5 to investigate the role of Cdk5 in vivo. These studies, along with organ-culture studies of whole eyes in the presence of a specific inhibitor of Cdk5, have shown that Cdk5 also regulates corneal epithelial wound closure in the intact eye. Current studies are investigating the relationship between Cdk5 and Src-family kinases in regulating corneal epithelial cell migration. Preliminary findings indicate that Src-family kinases are responsible for activation of Cdk5, which in turn, regulates the transport of activated Src from the perinuclear region to peripheral cell sites. In particular, Cdk5 regulates transport of Src to the leading edge of corneal epithelial cells in an in vitro scratch assay. To study this phenomenon in greater detail, we have developed an antibody to a site in the unique region of c-Src which is phosphorylated by Cdk5 and have generated a mutated form of c-Src which lacks this phosphorylation site. We are also exploring the role of Cdk5 in the lens. Our previous work has implicated Cdk5 in both cell-to-matrix and cell-to-cell adhesion in this tissue. To explore the mechanism of this effect we have constructed transgenic mice that overexpress Cdk5 or a kinase inactive form of Cdk5 specifically in lens fibers under the direction of the betaB1 crystallin promoter. Analysis of these animals will focus on the association of Cdk5 with other proteins known to be involved in cell-to-matrix and cell-to-cell adhesion. In collaboration with Dr. Sue Menko, we have already established that Cdk5 forms an in vivo complex with FAK, Src, and integrins in embryonic chicken lens fibers and differentiating epithelial cells. Studies with transgenic mice will attempt to extend these findings to a mammalian lens.